Background We are testing gene therapy, small molecule discovery, photoreceptor replacement and retinal reconstruction to identify novel modalities to treat retinal and macular degenerative diseases. All strategies are guided by the knowledge acquired through other projects of the molecular mechanisms of photoreceptor development, homeostasis, and disease (see EY000450). Gene therapy Retinal and macular neurodegeneration that are caused by loss-of-function mutations in a single gene are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in the RPGR or RP2 gene. In collaboration with Z. Wu (Gene Therapy Core, NEI) and T. Li (NNRL) we have completed pre-clinical studies in mouse models for the treatment of retinitis pigmentosa (RP) caused by mutations in these two genes. Experimentation is underway for Leber congenital amaurosis (LCA) caused by CEP290 mutations. Our group focused on animal models and genetic characterizations, and Z. Wu's group was instrumental on AAV-vector generation and took the lead for gene therapy. RPGR-XLRP- Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of XLRP and 15-20% of all inherited retinal degeneration, making it a target of wide applicability for gene therapy. We generated new stable adeno-associated virus (AAV)8 or AAV9 vectors carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-/- mice. Eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes (5). RP2-XLRP- We first characterized the Rp2-/- mouse in which early onset cone dysfunction, followed by progressive cone degeneration, mimics cone vision impairment in RP2 patients. The mice also exhibited distinct and significantly delayed falling phase of photopic electroretinogram (ERG) b -wave. A long-term efficacy study with an AAV vector that mediates stable RP2 protein expression in mouse photoreceptors showed preservation of cone function with wide dose range over 18-month duration. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression (3). In both RPGR-ORF15 and RP2 studies, the therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. However, retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in designing human trials. A broad treatment window and long-lasting therapeutic effects make both the RPGR-ORF15 and RP2 gene therapies attractive for clinical development. Even more so for RP2, which also displays a wide range of effective doses. A patent application describing these vectors has been submitted (4). CEP290-LCA- Experimentation is currently under way in a mouse model of LCA with the CEP290 AAV vector we recently designed (patent submitted, ref. 6). Use of iPSCs and ESCs for disease modeling and to develop therapies We are using both mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs) and in vitro cell-based cultures to develop disease models, biomarkers for cell replacement, and protocols for high throughput screens of small molecules. The studies are primarily focused on LCA caused by mutations in CEP290/NPHP6 or NPHP5 genes. Three-dimentional (3D) organoids generated from mouse and human ESCs and iPSCs are useful tools to study disease mechanisms and treatment paradigms. We have generated hESC lines expressing several fluorescent reporter proteins in retinal progenitors and precursors. We recently described H9 hESC subclone that carries a green fluorescent protein (GFP) reporter under control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors (1, 2). The CRXp-GFP reporter replicates endogenous CRX expression in vitro when cells are induced to form self-organizing 3D retina-like tissue. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted developmental sequence. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. Transcriptome profiling by RNA-seq of CRX+ developing photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina. However, many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation. Our studies established the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures and validating the pluripotent stem cell-derived 3D retina-like tissue as in vitro model for studies of the human retina. Several patient and control fibroblasts and iPSCs have been generated for CEP290-LCA, CEP290-Joubert Syndrome, NPHP5-LCA and NPHP5-Senior-Loken. These cell lines are being characterized to study the effect of gene mutation and of targeted therapies on photoreceptors. Small molecule screening We have characterized three different alleles of Cep290 in mice. Cep290/rd16 mice provide a good model for CEP290-LCA. We have generated ESC and iPSC lines from rd16 mouse that contained Nrl-GFP transgene to label developing and mature rods and generated 3D retinal organoids. We have optimized new protocols that produce 70%+ rods in wild type mouse stem cell-derived 3D retina and survive for as many as 35 days. The rd16 retinal organoids show abnormalities in rod differentiation and retinal architecture. We have initiated small molecule screening studies in collaboration with M. Swaroop and W. Zheng (NCATS) to facilitate rod maturation and survival using wild type retinal organoids and to rescue the phenotype using rd16 organoids. These proof-of-concept studies can be further evaluated on human cells at a later stage of therapy development. We are also generating transcriptomes of Nrl-GFP+ rods from wildtype and rd16 SC-derived retinal organoids to identify possible markers for disease progression and for developing rescue strategies. The retinal differentiation assay is also being employed to test the efficacy of AAV vectors encoding different domains of CEP290 for gene therapy. Rod or cone photoreceptors produced from hESCs or hiPSCs are a potential renewable source for cell-based therapy. However, lengthy differentiation protocols and low yield make their use currently unpractical. We are testing small molecule libraries using hES and hiPS cell lines with fluorescent reporter constructs driven by photoreceptor-specific promoters to identify compounds that facilitate the differentiation of high quality and pure transplantable photoreceptor cells with moderate cost and effort.